During the past fifteen years, Dr. Inouye's laboratory has elucidated the role of the N-terminal 77-residue propeptide in the folding of subtilisin, an alkaline serine protease. The propeptide functions as a single turnover catalyst, which is essential for the correct folding of the 275-residue protease domain of subtilisin. The propeptide is autoproteolytically cleaved and degraded to yield active subtilisin upon the completion of the fold process. An intramolecular domain such as the subtilisin propeptide, which is required for protein folding but not a part of the mature protein has been found in a number of proteins from bacteria to humans, and is termed "Intramolecular Chaperone (IMC)". The IMC-mediated protein folding provides a unique system to study the mechanism of protein folding in particular because a number of mutants within the propeptide have been isolated, which affects the folding process. Furthermore, Dr. Inouye's laboratory has found that some propeptide mutations cause the formation of stable conformers of subtilisin with different properties, and memorize structural alterations in the mutated propeptides. This phenomenon is called "protein memory". Elucidation of the molecular mechanism of protein memory is essential for our understanding of the basic principle of protein folding as well as a number of human diseases caused by protein misfolding. This proposal will focus on the following two specific aims:Molecular mechanisms of protein memory - Using the same method developed in Dr. Inouye's laboratory to crystallize the propeptide-subtilisin (S(221)C) complex, attempts will be made to obtain the mutant propeptide-conformer complex for crystallization and structural studies. Attempts will also be made to isolate other IMC mutations that cause altered subtilisin folding. Biochemical, biophysical and structural studies will determine the precise changes in structures causing protein memory. Identification and characterization of folding intermediates - The principal investigator's laboratory has successfully trapped a folding intermediate in a stable form by incorporating an S-S bridge. First a method will be established for a large-scale purification of the folding intermediate for structural studies, and biochemical and biophysical characterization. Attempt will also be made to isolate the crosslinked folding intermediates containing 'protein memory' mutations as another approach to study protein memory. Another emphasis is to investigate how the nascent folding intermediate can be activated with small peptide substrates.